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Abstract

Background

Vitamin E supplements containing tocotrienols are now being recommended for optimum
health but its effects are scarcely known. The objective was to determine the effects
of Tocotrienol Rich Fraction (TRF) supplementation on lipid profile and oxidative
status in healthy older individuals at a dose of 160 mg/day for 6 months.

Methods

Sixty-two subjects were recruited from two age groups: 35-49 years (n = 31) and above
50 years (n = 31), and randomly assigned to receive either TRF or placebo capsules
for six months. Blood samples were obtained at 0, 3rd and 6th months.

Results

HDL-cholesterol in the TRF-supplemented group was elevated after 6 months (p < 0.01).
Protein carbonyl contents were markedly decreased (p < 0.001), whereas AGE levels
were lowered in the > 50 year-old group (p < 0.05). Plasma levels of total vitamin
E particularly tocopherols were significantly increased in the TRF-supplemented group
after 3 months (p < 0.01). Plasma total tocotrienols were only increased in the >
50 year-old group after receiving 6 months of TRF supplementation. Changes in enzyme
activities were only observed in the > 50 year-old group. SOD activity was decreased
after 3 (p < 0.05) and 6 (p < 0.05) months of TRF supplementation whereas CAT activity
was decreased after 3 (p < 0.01) and 6 (p < 0.05) months in the placebo group. GPx
activity was increased at 6 months for both treatment and placebo groups (p < 0.05).

Conclusion

The observed improvement of plasma cholesterol, AGE and antioxidant vitamin levels
as well as the reduced protein damage may indicate a restoration of redox balance
after TRF supplementation, particularly in individuals over 50 years of age.

Background

Dietary and supplemental sources of vitamin E isoforms have been demonstrated to possess
unique properties that can influence critical pathways involved in cancer [1,2], cardiovascular [3,4] and neurodegenerative disease [5,6] development. Recent studies have identified tocotrienol, the lesser known isomer
of vitamin E as the more effective compound in providing such protection in comparison
to the well-established tocopherol [7-14]. The most promising function of tocotrienol has been reported in neuroprotection
[14,15] and stroke prevention with the latter being attributed to the lipid-corrective properties
of tocotrienol [11,13]. However, most data were generated from in vitro studies or by using animal models. To date, very limited data available from human
intervention study particularly involving supplemental vitamin E and its fundamental
effects on diseases and ageing.

Recent interest has focussed on finding compounds that could intervened the chemical
processes underlying age-related degenerative diseases in which has been demonstrated
to be also responsible for the ageing phenomena [16,17]. Although vitamin E supplementation has yielded equivocal results in human intervention
studies largely on a short term basis [18-20], supplementation studies measuring benefits to cardiovascular end points are still
controversial with some showing no benefits and some even detrimental effects [21-23]. Studies are warranted to elucidate the underlying mechanism on the effects of vitamin
E in preventing or treating these diseases that may explain the observed effects.

Most vitamin E supplements available in the market usually contain only alpha tocopherol.
However, recently with the availability of tocotrienols commercially, the uses of
tocotrienol-containing supplement are more widespread. There has been a paradigm shift
to vitamin E supplementation where all isomers of vitamin E are recommended rather
than alpha tocopherol alone [24]. However, it has yet to be determined whether supplementation with a mixture of vitamin
E isomers containing high tocotrienol fractions will affect biochemical parameters
and other blood indices and the possible underlying mechanism. We are interested in
investigating age-associated biochemical changes with tocotrienol enriched vitamin
E supplementation and identifying the specific oxidation pathways involved.

Different aged individuals respond differently to various food and vitamin supplementation
[25]. Needs for supplement may be different with the different age where older people
may benefit from supplementation as they were reported to have lower antioxidant levels
[26].

We previously reported that TRF supplementation decreased DNA damage in healthy older
adults, mostly in those over 50 years of age, and that the levels of damage were associated
with age [27]. In the present work, changes in lipoprotein-lipid profile, protein carbonyl content,
advanced glycosylation end products (AGEs), malondialdehyde (MDA) levels, levels of
the antioxidant vitamins E and C, and antioxidant activities of superoxide dismutase
(SOD), catalase (CAT) and glutathione peroxidase (GPx) were measured in a randomized,
double-blinded, placebo-controlled intervention study of TRF supplementation. We also
studied the association between oxidative biomarkers and antioxidant levels to further
elucidate the oxidant-antioxidant balance with increasing age.

Methods

Study design

Healthy volunteers were recruited through screening of a population study on oxidative
stress and ageing. Selected individuals were aged 35 years and older, non-smokers,
not pregnant, not taking any vitamin supplements or minerals, alcohol, or drugs, and
free of cardiac, hepatic, renal or any other chronic diseases. Adult females were
recruited as the compliance from this gender is higher and mostly are non-smokers.
Results of a pre-study consisting a full physical examination, previous medical history,
blood chemistry and haematology were used to confirm suitability. Sixty-two subjects
were recruited from two age groups, 35-49 years (n = 31) and over 50 years (n = 31),
and randomly assigned to receive either Tocotrienol Rich Fraction (TRF) capsules (160
mg/day) daily in a single evening dose or an identical placebo for a period of six
months. All subjects were requested to consume the capsules after dinner to ensure
proper absorption [28,29] and encouraged to maintain their usual lifestyle throughout the study period. The
commercially prepared TRF (Tri E® Tocotrienol) is a palm-based vitamin E consisted of approximately 74% tocotrienols
and 26% tocopherol in soft gelatine capsules containing palm superolein oil, and was
supplied by Sime Darby Bioganic Sdn. Bhd. (previously known as Golden Hope Bioganic,
Selangor, Malaysia). Each capsules contained approximately 70.4 mg α-tocotrienol,
4.8 mg β-tocotrienol, 57.6 mg γ-tocotrienol, 33.6 mg δ-tocotrienol and 48 mg α-tocopherol.
The capsules given throughout the study were from the same lot and provided loosely
in plastic containers. The placebo capsules contained only palm superolein oil. In
addition to plasma samples for vitamin E levels, compliance was assessed by capsule
counts at each 3-month interval. The amount of allotted and returned capsules for
each participant is recorded during the interval visits. The treatment was double
blinded throughout the study period until all data were collected, after which the
randomisation code was broken. The protocol of the study was approved by the Research
and Ethics Committee of the Faculty of Medicine, Universiti Kebangsaan Malaysia. Written
informed consent was obtained. Subject demographics are summarised in Table 1.

Table 1. Baseline and intervention characteristics of the study groups

Sample collection

Blood sampling was performed at baseline (month 0), 3 months and 6 months of supplementation.
Venous blood samples were drawn from fasting subjects into lithium heparin-coated
and K2EDTA-containing tubes (BD Vacutainer, Becton, Dickinson and Company, Franklin Lakes,
NJ, USA) for plasma extraction while into a plain tube for serum extraction. Plasma
and serum were immediately separated by centrifugation at 3000 g for 10 minutes. The
obtained packed erythrocytes were washed three times with 0.9% sodium chloride solution.
Heparinized plasma aliquots were separated for lipid profiling as well as vitamin
C and vitamin E determination. Plasma-EDTA aliquots were used for protein carbonyl
and MDA quantification whereas serum aliquots were used for AGE product testing. Washed
erythrocytes were used for determination of antioxidant activities of SOD, CAT and
GPx. Plasma samples for vitamin C determination were deproteinised with 5% perchloric
acid and centrifuged at 1000 g for 2 minutes, and the resulting clear supernatant
was transferred into new tubes. Lipid profile testing was done immediately whereas
other samples were frozen at -80°C until further analysis.

Plasma vitamin E determination

Plasma tocopherol and tocotrienol were determined by HPLC. Briefly, stored plasma
samples were thawed and aliquots of 200 µl plasma and 50 µl 95% ethanol containing
10 µg/ml butylated hydroxytoluene (Sigma Chemical Co., St. Louis, MO, USA) were pipetted
into tubes, covered and mixed vigorously for 5 seconds. 1 ml of absolute ethanol (Merck
KGaA, Darmstadt, Germany) was then added to each tube. The tubes were covered and
mixed again before being centrifuged at 1500 g for 15 minutes at 18°C. The bottom
pellet was carefully removed with a spatula. Hexane, 3 ml (Merck KGaA, Darmstadt,
Germany) were then added to each tube and mixed vigorously for 5 minutes. The samples
were then centrifuged at 1500 g for 15 minutes at 18°C. After centrifugation, a portion
of the upper layer (2.5 ml) was carefully removed, placed in new tubes and vacuum-evaporated
for 40 minutes. The dried sample residue was then reconstituted in 100 µl HPLC grade
hexane (Merck KGaA, Darmstadt, Germany), vortexed, and passed through a 0.45 µm filter
to remove any non-dissolved particles. The samples were then transferred to amber
vial inserts with care taken to avoid air bubbles and analysed by HPLC using a Shimadzu
RF-10A XL fluorescence detector (Shimadzu Corporation, Kyoto, Japan) at an excitation
wavelength of 294 nm and an emission wavelength of 330 nm. Isomers α-tocopherol, γ-tocopherol,
α-tocotrienol, γ-tocotrienol and δ-tocotrienol were separated on a 250 mm × 4.6 mm,
5 µm Allsphere Silica column (Alltech Associated, Inc, IL, USA) and eluted with a
mobile phase of 99:1 (v/v) hexane-isopropanol at a flow rate of 1.5 ml per minute.
The identity of each compound was confirmed by co-elution with spiked standard. All
external standards used were obtained from Malaysian Palm Oil Board (MPOB), Malaysia.
The peaks were quantified and integrated with Shimadzu Class-VP™ version 6.1 LC Workstation
software (Shimadzu Corporation, Kyoto, Japan).

Plasma vitamin C determination

The measurement of plasma vitamin C (ascorbic acid) was performed using HPLC as described
by Pachla and Kissinger [30]. Plasma ascorbic acid was chromatographically separated on a 25 cm VYDAC® Genesis C-18 4 µm column (Grace Davison, USA) using a mobile phase containing 0.04
M sodium acetate, 0.48 mM disodium EDTA, 2.9 mM tetrabutylammonium hydroxide and 1.4%
methanol at pH 4.75. Detection was performed on an electrochemical detector (Gilson
model 142, Gilson Medical Electronics S.A., Villiers-le-Bel, France) set at an applied
potential of +700 mV and referenced to an Ag/AgCl electrode, with a flow rate of 1
ml/min and sensitivity of 100 nA/V on a Gilson HPLC set (Gilson Medical Electronics
S.A., Villiers-le-Bel, France). Assay calibration was performed for each run using
six concentrations of the calibrator (0, 2, 4, 6, 8, 10 and 12 mg/L). Stored deproteinised
plasma samples were thawed, added with 5 mg/L 3,4-dihydroxybenzylamine (Sigma Chemical
Co., USA) and spiked with 5 mg/L ascorbic acid (Sigma Chemical Co., St. Louis, MO,
USA). 3,4-dihydroxybenzylamine was added to all samples and calibrators as an internal
standard whereas uric acid Raichem™ (Reagents Applications Inc., USA) was injected
separately for peak identification. All samples were filtered through a 0.45 µm membrane
before being subjected to HPLC and all peaks were baseline separated. Ascorbic acid,
3,4-dihydroxybenzylamine and uric acid peaks were resolved and detected within 11
minutes of run time and were identified using the retention times of calibrators.
Peaks were integrated using peak-area ratios of ascorbic acid to 3,4-dihydroxybenzylamine.

Plasma protein carbonyl determination

Carbonyl content in oxidatively-modified proteins was assayed using the Cayman Chemical
protein carbonyl assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) based on
the method of Levine et al. [31] by following the manufacturer instructions. Briefly, 2,4-dinitrophenyl-hydrazine
(DNPH) reacts with protein carbonyls, forming a Schiff base to produce the corresponding
hydrazone. The protein-hydrazone produced was quantified at an absorbance of 370 nm
with an extinction coefficient of 22,000 M-1cm-1. Each sample was assayed with a parallel control and the concentration of carbonyls
was determined after correction with the respective control. The carbonyl content
was then standardized against the protein concentration in the sample and expressed
as nmol carbonyl per mg protein. The amount of protein was calculated from a bovine
serum albumin (Sigma Chemical Co., St. Louis, USA) standard curve (0.25-2.0 mg/ml)
read at 280 nm.

Serum AGE determination

Serum advanced glycosylation end products (AGEs) was measured with an in-house competitive
enzyme immunoassay technique developed by Wan Nazaimoon and Khalid [32]. Briefly, microtitre wells were coated with AGE-BSA at 8 µg/ml followed by an overnight
incubation with 20 µl of the prediluted sample (1:6) and 80 µl anti-AGE-KLH (1:8000).
HRP-labelled goat anti-rabbit (1:3000) was used as the secondary antibody and 3,5',5,5'-tetramethylbenzidine
dihydrochloride (Sigma Chemical Co., St. Louis, USA) as the substrate. The colour
reaction was stopped with 1.25 M sulphuric acid and the absorbance was read at 450
nm with reference at 620 nm. All samples were assayed in triplicate and assay performance
was monitored using a set of in-house quality control sera containing three different
levels of AGE.

Plasma MDA determination

Plasma malondialdehyde (MDA) was determined by HPLC based on the derivatisation of
MDA with 2,4-dinitrophenylhydrazine (DNPH) (Sigma-Aldrich, St. Louis, MO, USA) as
described by Pilz et al. [33] with some modifications. Briefly, stored plasma samples were thawed and aliquots
of 250 µl plasma were mixed with 50 µl 6 M sodium hydroxide (Merck, Germany). The
sample mixture was incubated at 60°C for 30 minutes in a water bath. After cooling
to room temperature, the hydrolysed sample was then acidified with 125 µl 35% (v/v)
perchloric acid to precipitate proteins and centrifuged at 6000 g for 10 minutes at
18°C. The supernatant was transferred into fresh tubes and the sample was then derivatised
with 50 µl 5 mM DNPH for 30 minutes at room temperature. Derivatised samples were
used for HPLC analysis and protected from light from this step onwards.

Analytical HPLC separations were performed on a Shimadzu Chromatographic system (Shimadzu
Class-VP™ version 6.1 LC Workstation software, Shimadzu Corporation, Kyoto, Japan)
with a diode array detector equipped with an auto injector and operated at 310 nm
on a 150 mm × 3.9 mm, 5 µm alphaBond C18 column (Alltech Associated, Inc. IL, USA).
Samples and standards were eluted with a mobile phase consisting of 380 ml acetonitrile
with 620 ml of distilled water, acidified with 0.2% (v/v) acetic acid and degassed
at a flow rate of 0.6 ml/min. The plasma MDA level was calculated from a calibration
curve prepared by acidic hydrolysis of 1,1,3,3-Tetraethoxypropane (TEP) (Sigma-Aldrich,
St. Louis, MO, USA).

Determination of erythrocyte antioxidant enzymes

SOD, CAT and GPx activities in the hemolysates were expressed as U/mg Hb. Erythrocyte
Cu, Zn-SOD activity was assayed using the spectrophotometric indirect inhibition technique
of Beyer and Fridovich, which is based on the ability of SOD to inhibit the photoreduction
of nitro blue tetrazolium [34]. CAT activity was measured in erythrocytes using the method of Aebi [35] with hydrogen peroxide as the substrate. This method is based on the decomposition
of hydrogen peroxide and measured by decreased absorbance at 240 nm. GPx activity
was measured in erythrocytes using the coupled method of Paglia and Valentine with
t-butyl hydroperoxide as the substrate [36]. Hemoglobin was assayed using the cyanmethemoglobin procedure (Eagle Diagnostics,
Desoto, Texas, USA) based on the determination of cyanmethemoglobin at 540 nm.

Statistical Analysis

Statistical analysis was performed using Statistical Package for Social Sciences (SPSS)
Version 11.5 (Chicago, Illinois, USA). Mixed model analysis of variance (ANOVA) was
used to compare changes from baseline to 3 and 6 months for all variables to ascertain
the effects of treatment. One way analysis of variance was used to identify significant
differences between age groups. The associations of all parameters were assessed by
partial correlations and analyses of covariance to account for the increased proportion
of women in the study group. Pearson's correlation coefficient was determined for
baseline variables to determine their relationships. Mauchly's test of sphericity
was used to assess the homogeneity of variance and Dunnett's test was performed to
compare the means of the treatment group to the means of the control group. Bonferroni's
adjustment was applied to control the inflation of Type 1 error across multiple tests.
All data are presented as mean

+

standard error of the mean (SEM). The null hypothesis was tested using a 2-tailed
α < 0.05 criterion. PS Program version 2.1.31 was used to determine the power of study
in which the probability of rejecting the null hypothesis with Type I error was set
at α = 0.05, given the specified sample size n = 30, a standard deviation σ = 0.104
and when the true difference in population means was δ = 0.097. The statistical power
obtained from our approach was 0.9977.

Results

Subject characteristics

The baseline characteristics of subject age, gender, blood pressure, pulse, body mass
index and fasting blood glucose in the TRF groups were similar to those in the placebo
groups (Table 1). Systolic and diastolic blood pressure in the younger group (35-49 years old) were
significantly lower than those in the older group (over 50 years old) while no significant
differences was observed between groups regarding to pulse, body mass index (BMI)
or fasting blood sugar (FBS). None of these clinical parameters showed statistical
difference upon supplementation except for BMI (Table 1) where the younger group who received TRF demonstrated reductions in BMI at 3 months
(p = 0.024) and 6 months (p < 0.001).

Lipid profile

Plasma total cholesterol was within the normal range in all subjects before supplementation.
There was no significant difference in lipids level between the younger and older
groups at baseline (Table 2). A statistically significant effect for duration of treatment was observed in HDL-C
in TRF group (F = 4.196, p = 0.020, effect size = 0.139, power = 0.713). The HDL-C
level in the younger group increased significantly after 6 months of Tocotrienol supplementation
(p = 0.014) as compared to the baseline, while in the > 50 year-old group, a non-significant
elevation of 8% was observed. However, the plasma ratio of HDL-C to total cholesterol
improved in both younger (p = 0.007) and older groups (p = 0.029) with supplementation.
Values obtained for all lipid variables were within the normal range.

Overall, lipid-corrected tocotrienol levels were significantly increased in TRF supplemented
subjects when compared to baseline and placebo-receiving subjects after 6 months (F
= 10.068, p = 0.005, effect size = 0.324, power = 0.857). However, further increases
in tocotrienol levels were only observed in the > 50 year-old group after 6 months
of treatment (F = 11.197, p = 0.006, effect size = 0.483, power = 0.866). Regardless
of the treatment type, levels of lipid-corrected total tocotrienols in the older group
were lower than in the younger group. With treatment, however, levels of lipid-corrected
total tocotrienols in older subjects approached levels observed in younger subjects.

Plasma vitamin C concentrations increased in both groups regardless of the treatment
received. Further analysis of age groups showed similar increases for all groups to
varying degrees of significance.

Antioxidant enzymes

A significant effect for duration was observed in SOD activity with TRF treatment
(F = 6.838, p = 0.006, effect size = 0.229, power = 0.826) as well as in CAT and GPx
activity with placebo (F = 4.185, p = 0.002, effect size = 0.160, power = 0.707 and
F = 5.271, p = 0.009, effect size = 0.193, power = 0.809 respectively). SOD activity
decreased after 3 and 6 months of TRF treatment (Table 5); these effects were seen in both younger and older groups, although only the > 50
year-old group reached statistical significance (Table 6). CAT activity was decreased in the placebo group after 3 months; this change was
also only observed in the > 50 year-old group. Similar effects were observed for GPx,
where only subjects in the > 50 year-old group showed increased activity after 6 months
regardless of treatment.

Table 6. Erythrocyte antioxidant enzymes activity according to age group

There was nearly no association between SOD activity and age (Figure 1), whereas GPx activity slightly declined with age (Figure 2), while CAT activity slightly increased with age (Figure 3); however, none of these correlations was significant. Supplementation with TRF strengthened
the relationship between SOD activity and age as well as for CAT activity. GPx activity
was reversed in the older individuals in association with age.

Oxidative markers

Changes in protein carbonyl levels were statistically significant with duration of
treatment (F = 6.193, p = 0.008, effect size = 0.212, power = 0.810). Protein carbonyl
content was significantly decreased after 6 months of TRF treatment (p = 0.002) as
compared to baseline (0 month) (Table 7). Further grouping by age (Table 8) revealed a marked reduction (p < 0.001) in the > 50 year-old group after 6 months
of treatment. Nonetheless, no significant effect for duration was obtained in AGE
and MDA levels despite both levels were reduced in the older TRF-treated group after
3 months and remained low thereafter; however, this tendency did not reach significance.

The relationships between age and oxidative stress marker levels are shown in Figures
4, 5 and 6. All measured biomarkers (protein carbonyl, AGE and MDA) were weakly correlated with
age. TRF treatment reversed these relationships, particularly for MDA, which showed
a correlation coefficient of close to 0.4 (p < 0.05).

Discussion

Human ageing is affected by both genetic factors and lifestyle-related factors such
as diet. Dietary intervention is feasible, as nutrients can affect the rate of ageing
by altering the type and quantity of proteins synthesized [37] by modulating gene expression [38], thereby altering the oxidative status of individuals [39].

Our results indicate that daily supplementation for up to 6 months with TRF raised
plasma HDL cholesterol levels as early as 3 months, thereby increasing the HDL-cholesterol/total
cholesterol ratio. This ratio reflects the proportion of anti-atherogenic to atherogenic
lipids and has been suggested as a better predictor of cardiovascular disease risk
than the individual lipoprotein values [40]. TRF might thus help to reduce the risk of coronary heart disease (CHD) in healthy
older adults. In fact, HDL cholesterol increases of the magnitude observed in this
study have been associated with a 22.5% reduced risk of cardiovascular events [41]. Raising plasma HDL cholesterol and thus the HDL-cholesterol/total cholesterol is
recommended by the American Diabetes Association (ADA) guidelines together with lowering
plasma triglycerides for high-risk individuals particularly older adults as major
mortality cases of CHD were 65 years old or older [42].

Conflicting data have been reported regarding effects of vitamin E supplementation
which were mainly α-tocopherol on HDL cholesterol. Increased HDL cholesterol after
α-tocopherol supplementation has been reported by some investigators [43-45] and disputed by others [46,47]. It should be noted that the supplement used in this study was high in tocotrienols,
which has been reported to have different effect from α-tocopherol [48]. Tocotrienol but not tocopherol increases HDL cholesterol by inhibiting HMG-CoA reductase
through signalling, thereby regulating cholesterol biosynthesis [49]. Tocotrienol may increase HDL in this study by modulating signal transduction and
gene expression; specifically, and may normalize any aberrant gene expression incurred
by aging [50]. Increases in HDL could be attained by increasing physical exercise [51,52] but similar effects by supplementation of vitamins have not been reported in human.

Compliance of the subjects was indicated by the observed increase in plasma lipid-corrected
total tocotrienol and tocopherol concentration. Standardization of plasma vitamin
E levels to total cholesterol was necessary to control for age-related changes in
baseline cholesterol levels as vitamin E is transported by the lipoproteins. The finding
that tocotrienol levels were increased is of particular interest, as tocotrienol is
now reported to have functions distinct from α-tocopherol as reviewed by Sen et al. [53]. The marked increase in tocotrienol in the > 50 age group is interesting and suggests
an increased bioavailability and possibly the need for tocotrienol with aging. The
level of total tocotrienol was slightly lower in the older adults as opposed to the
younger group. Supplementation of older subjects with TRF restored plasma vitamin
E availability to near the levels of in the controls of the younger group. We speculate
that a steady state plasma vitamin E concentration was achieved after 6 months of
supplementation, as plasma concentrations were similar to those in the younger group
were seen at that time point. Some studies have reported the achievement of steady-state
plasma vitamin E levels after 10 to 15 days of supplementation with either natural
or synthetic forms of α-tocopherol at much higher dosages [54-56]. Considering the well-documented preferential absorption and transportation of different
vitamin E isomers in the body and by taking into account the tocotrienol-rich composition
in the TRF, such a slow but steady increment is reasonable.

The tocotrienols are found in a wide variety of foods and it has been suggested recently
that all 8 isomers of vitamin E may be necessary for optimum health [24]. This requirement maybe more crucial for the older individuals where digestion and
absorption may not be as efficient resulting in the potential benefits of supplementation.
Differences noted in the plasma levels of tocotrienols detected in various human studies
are mainly due to the different daily fat diet. Asians consume higher levels of palm
oil rich in tocotrienols. Therefore, a comparatively higher absorption of tocotrienol
and thus better level found in circulation.

The elevated plasma vitamin C level detected in the present study was most likely
absorbed from the diet, as the supplement is not a source of vitamin C. Indeed, we
found a positive correlation between vitamin C intake and plasma vitamin C levels
(r = 0.308, p = 0.048). This is in accordance with findings by Padayatty et al. [57] who had reported that small changes in oral intake of vitamin C resulted in large
changes in plasma vitamin C concentration. The increase in plasma vitamin C might
also result from a complementary effect by vitamin E in an interlinking antioxidant
network. The involvement of vitamin C in regenerating vitamin E directly from its
tocotrienoxyl or tocopheroxyl radical back to tocotrienol and tocopherol respectively
has been well documented [58]. Increased levels of vitamin E might reflect increased reactions with reactive free
radicals, additional formation of tocotrienoxyl or tocopheroxyl radical, and a further
increased need for vitamin C.

The variation observed in enzyme activity might have been due to the different roles
of the analysed antioxidant enzymes. The functional roles of these enzymes are well
established, with SOD acting upstream by dismutating reactive superoxide anion radicals
into more stable hydrogen peroxide (H2O2) whereas catalase and GPx function downstream by converting H2O2 into water and oxygen in apparently parallel pathways. Changes in antioxidant enzymes
activity observed were clearly in favour of the > 50 years old group. Reduction in
SOD activity with TRF supplementation after 3 and 6 months in the older group possibly
due to lesser formation of radicals as a result of radical scavenging effect by tocotrienol
and tocopherol. On the other hand, increase in GPx activity in both placebo and TRF
group after 6 months possibly due to higher need for H2O2 removal. Increased level of tocotrienol and tocopherol attained by TRF supplementation
might reflect more radical scavenging activity, followed by increased H2O2 formation and therefore increased requirement for its removal. As for the placebo
group, increased intake of dietary vitamin C might results in a similar increase in
radical scavenging activity and the subsequent reactions involving formation and detoxification
of H2O2. Given a decline in the catalase activity in the placebo group, the action of removing
H2O2 was predominantly done by GPx. In the current study, the enzymes activity measured
was evidently influenced by TRF supplementation as shown by the shifted correlation
patterns with age.

Long term supplementation of TRF for 6 months caused the increase in plasma vitamin
E availability (both tocopherol and tocotrienol) observed in the current study, accompanied
by changes in the oxidative stress biomarkers measured. Protein carbonyls have been
described as oxidized amino acids resulting from direct oxidation of protein by reactive
oxygen species [59]. Proteins are also modified indirectly by glycation or glycoxidation of amino groups
with the eventual formation of the advanced glycosylation end products (AGEs) [60]. Consistent with the fall in plasma concentrations of carbonylated protein with TRF
supplementation, a sharp decrease in serum AGE was observed. When the effect of age
was factored out of the statistical model, it was found that the interaction between
supplementation and duration was significant for the older individuals, indicating
a favourable gain in the older group. Figures 4, 5 and 6 give individual presentations of the changes in these oxidative markers with age.
Ascending trends of protein damage and lipid peroxidation accumulation during ageing
as shown by the correlative data were reduced, even reversed by TRF supplementation.
These findings confirm that nutritional intervention can exert cumulative effects
on oxidative stress in healthy individuals in the long term [27]. The unique combination of vitamin E isomers used in the study might have acted synergistically
to provide the beneficial effect.

The reduced levels of oxidative markers were mainly observed in the older group, for
whom the lower cut-off point was 50 years of age. This is of interest as previous
studies typically evaluated older subjects, mostly 60 years old and over. It is also
noteworthy that in the present study the treatment was administered to healthy individuals
for a lengthy period of 6 months and studies involving supplementation of this duration
are fairly limited. This may then results in the observed changes in oxidative status
as measured by protein carbonyl and AGE. Although some of the younger subjects in
the study also showed increased levels of antioxidant vitamins, the magnitude of changes
was less evident as compared to the older individuals. The absence or lack of response
by the younger age group might reflect a well-maintained antioxidant level, more effective
maintenance of oxidative balance, and better defence against spontaneous oxidative
injury. It is thus possible that TRF supplementation did not provide any further improvement.
Although baseline antioxidant levels in the > 50 year-old group were similar to those
in the < 50 year-old group, baseline oxidative marker levels were higher in the older
group, suggesting a higher level of oxidative damage. However, the amount of damaged
lipids and proteins in this group was reduced by supplementation, probably due to
the increased requirement for antioxidants in older individuals. It is possible that
an antioxidant threshold for optimum performance exists and that this threshold (and
therefore the requirement for antioxidants) could increase with ageing, thus allowing
supplementation to generate an effect in the present study. A long-term prospective
study will be required to test this hypothesis, particularly at the molecular level.

Compelling evidence suggests a new level of action for vitamin E under the non-antioxidative
control in protection against disease [50]. Therefore, TRF might not only act directly or solely as an antioxidant, but it may
actually also act through signalling pathways and specific signal-regulated protein
reaction as suggested by Sen et al. [14,61]. Tocotrienol was shown to provide complete neuroprotection via antioxidant-independent
mechanism with the protective property reported not only limited in response to non-oxidative
challenges but also to oxidative insults [14,15]. Further studies of the effects of tocotrienols in a cell model are currently underway
in our laboratory.

Conclusion

Our data revealed an age-related increase in oxidative damage. We established a role
of nutritional supplementation in oxidative damage and antioxidant levels in older
individuals. To our best knowledge, tocotrienol-rich vitamin E supplementation has
not yet been studied in relation to oxidative stress in healthy older individuals.
Consistent with increased concentrations of plasma antioxidants (vitamins E and C),
we observed significant decreases in protein carbonyl and AGE levels, as well as improvement
of plasma cholesterol levels. The protective effects of TRF supplementation observed
in this study might represent a restoration of redox balance, particularly in the
> 50-year old group. Increased tocotrienol level might be an important mechanism by
which TRF supplementation confers its protective benefits via protection against oxidative
stress, involvement in oxidized protein repair, besides contributing to the regulation
of redox homeostasis through signalling.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SFC was involved in the acquisition, analysis and interpretation of data in addition
to drafting the manuscript; JI, NAAH, AAL, ZZ and AAK made significant contributions
to the acquisition of the data, SM was involved in interpretation of data and critical
analysis of intellectual content of the manuscript, MM and YAMY contributed to the
design, acquisition and interpretation of the data; and WZWN was instrumental in the
study's inception, design and approval while providing critical analysis of data interpretation
and manuscript review. The final manuscript have been read and approved by all authors.

Acknowledgements

The authors thank Ms. Zalina Hamid from Sime Darby Bioganic Sdn. Bhd. (Selangor, Malaysia)
for supplying the TRF and placebo capsules used in the study, and Dr. Wan Nazaimoon
Wan Mohamud (Division of Endocrinology, Institute for Medical Research, Kuala Lumpur,
Malaysia) for kindly providing us with techniques and antibodies to measure the serum
AGE concentration. Financial support for this study was provided by grants awarded
from the Malaysian Government, the IRPA 06-02-02-0016-PR0008/09 and UKM-GUP-SK-07-21-199.